Nucleic acid-containing composition, preparation and use thereof

ABSTRACT

Pharmaceutical composition useful for transfecting a nucleic acid and characterised in that it contains, in addition to the nucleic acid, at least one transfecting agent and a compound causing the condensation of the nucleic acid, wherein the compound is totally or partly derived from a histone, a nucleoline, a protamine and/or a derivative thereof. The use of the composition for transferring nucleic acids in vitro, ex vivo and/or in vivo is also described.

This application is a continuation of U.S. application Ser. No.08/894,339, filed Aug. 15,1997, now U.S. Pat. No. 5,945,400 which wasthe National Stage of International Application No. PCT/FR96/00248,filed Feb. 15,1996.

The present invention concerns the field of gene therapy and relatesmore particularly to the in vitro, ex vivo and/or in vivo transfer ofgenetic material. The invention proposes in particular a novelpharmaceutical composition which is useful for efficiently transfectingcells. The invention also relates to the uses of this composition.

Chromosomal deficiencies and/or anomalies (mutation, aberrantexpression, and the like) are the cause of many diseases, of hereditaryor non-hereditary nature. Conventional medicine has for a long timeremained powerless as far as they are concerned. Nowadays, with thedevelopment of gene therapy, it is hoped to be able from now on toprevent or correct this type of chromosomal aberration. This novelmedication consists in introducing genetic information into the affectedcell or organ, for the purpose of correcting this deficiency or anomalytherein or alternatively for the purpose of expressing a protein oftherapeutic value therein.

The main obstacle to the penetration of a nucleic acid into a cell ortarget organ lies in its size and the polyanionic nature of this nucleicacid, which oppose its passage across cell membranes.

In order to relieve this difficulty, various techniques are nowadaysproposed including, more particularly, the transfection of naked DNAacross the plasma membrane in vivo (WO90/11092) and the transfection ofDNA via a transfection vector.

As regards the transfection of naked DNA, the efficacy of this stillremains very low. Naked nucleic acids possess a short half-life inplasma on account of their degradation by enzymes and their removal viaurinary routes.

Regarding the second technique, this also proposes two strategies:

The first uses natural transfection vectors, namely viruses. It is thusproposed to use adenoviruses, herpes-viruses, retroviruses and, morerecently, adeno-associated viruses. Although these vectors prove to beof high performance as regards transfection, it is unfortunately notpossible to exclude as far as they are concerned certain risks ofpathogenicity, replication and/or immunogenicity, which are inherent totheir viral nature.

The second strategy consists advantageously in using non-viral agentscapable of promoting the transfer and expression of DNA in eukaryoticcells.

The subject of the present invention is directed more particularlytowards this second strategy.

Chemical or biochemical vectors represent an advantageous alternative tonatural viruses, in particular for this absence of viral recombinationand/or immunological response. They have no pathogenic power, there isno risk of multiplication of the DNA within these vectors and there isno theoretical limit associated therewith as regards the size of the DNAto be transfected.

These synthetic vectors have two main functions, to condense the DNA tobe transfected and to promote its cell binding as well as its passageacross the plasma membrane and, where appropriate, the two nuclearmembranes.

On account of its polyanionic nature, DNA naturally has no affinity forthe plasma membrane of cells, which membrane is also polyanionic. Inorder to overcome this drawback, non-viral vectors generally all havepolycationic charges.

Among the synthetic vectors developed, cationic polymers of polylysineand DE dextran type or alternatively cationic lipids or lipofectants arethe most advantageous. They have the property of condensing DNA and ofpromoting its association with the cell membrane. More recently, theconcept of targeted transfection mediated by a receptor has beendeveloped. This technique exploits the principle of condensing DNA, byvirtue of the cationic polymer, while at the same time directing thebinding of the complex to the membrane using a chemical coupling betweenthe cationic polymer and the ligand of a membrane receptor present atthe surface of the cell type which it is desired to graft. Screenings ofthe receptor with transferrin and insulin, and screening of thehepatocyte asialoglycoprotein receptor have thus been described.

However, the synthetic vectors proposed to date are still far fromgiving as good performance as viral vectors. This could be theconsequence of insufficient condensation of the DNA to be transfectedand/or difficulties, encountered by the transfected DNA, of leaving theendosome and penetrating into the cell nucleus. Lastly, other drawbacksare directly associated with the nature of the cationic polymers of thelipofectants used.

The subject of the present invention is precisely to propose anadvantageous solution to these problems.

More precisely, the present invention relates to a pharmaceuticalcomposition which is useful for the transfection of a nucleic acid,characterized in that it contains, besides the said nucleic acid, atleast one transfection agent and a compound involved in the condensationof the said nucleic acid; the said compound being derived, partly ortotally, from a histone, a nucleoline, a protamine and/or one of thederivatives thereof.

The Applicant has discovered, unexpectedly, that the presence of such acompound within a transfecting composition based on a standardtransfection agent made it possible to reduce considerably the amount ofthis agent, with the beneficial toxicological consequences stemmingtherefrom, without bringing any prejudice to bear on the transfectingactivity of the said composition. On the contrary, this compositionadvantageously has a higher level of transfection.

In the sense of the invention, a compound involved in the condensationof the nucleic acid covers any compound which directly or indirectlycompacts the nucleic acid. More precisely, this compound may either actdirectly on the nucleic acid to be transfected or may be involved at thelevel of an associated compound which itself is directly involved in thecondensation of this nucleic acid.

Preferably, it acts directly on the nucleic acid.

According to a specific embodiment of the invention, the compoundinvolved in the condensation of the nucleic acids consists, totally orpartly, of peptide units (LysThrProLysLysAlaLysLysPro) SEQ ID No. 1and/or (AlaThrProAlaLysLysAlaAla) SEQ ID No. 2 or one of theirderivatives, it being possible for the number of units to range between2 and 10. In the structure of the compound according to the invention,these units may be repeated continuously or non-continuously. Thus, theymay be separated by connections of biochemical nature, for example oneor more amino acids, or of chemical nature.

The particular choice, a compound according to the invention, of apeptide or pseudopeptide possessing a majority of amino acids with basicnature, such as lysine, histidine or arginine, is particularlyadvantageous in the context of the present invention. This compound mayalso possess a β-sheet conformational structure. Basic amino acids are,indeed, more specifically involved in peptide-nucleic acid bonding. Theyparticipate in the establishment of ionic hydrogen bonds between the twospecies, thus promoting the condensation of the nucleic acid. As regardsthe 8-sheet structure, this is characterized by better accessibility ofthe majority of the carbonyl bonds and of the hydrogen atoms which, onaccount of their respective acceptor and donor natures, also favour theformation of bonds with the nucleic acid to be compacted.

Such a compound is more preferably all or part of a histone, anucleoline, protamine and/or one of the derivatives thereof.

Histones and protamines are cationic proteins which naturally compactDNA. They are thus responsible in vivo for the condensation ofnon-transcribed DNA and the DNA of certain viruses. As histones whichmay be used in the context of the present invention, mention may be mademore particularly of histones H1, H2a, H3 and H4. As regards nucleoline,this is a nucleolar protein which would appear to possess a synergisticeffect with respect to the histone H1 during the condensation of DNA bythe latter. In the context of the present invention, the compound mayadvantageously be represented by a peptide sequence derived from theN-terminal part of nucleoline, and more precisely corresponding to thesequence (AlaThrProAlaLysLysAlaAlaAlaThrProAlaLysLysAlaAla) (COOH)(SEQID No. 3).

Preferably, the compound used according to the invention is a sequencederived from histone H1 and more preferably from its C-terminal domainand more particularly corresponds to the sequence(LysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro) (COOH) (SEQ IDNo. 4).

As an illustration of this family of compounds according to theinvention, mention may also be made of the following oligopeptides:

AlaThrProLysLysSerAlaLysLysThrProLysLysAlaLysLysPro (COOH). (SSEQ ID No.5) and

LysLysAlaLysSerProLysLysAlaLysAlaAlaLysProLysLysAlaProLysSerProAlaLysAlaLysAla(COOH). (SEQ ID No. 6).

As regards more particularly the sequences derived from protamines whichcan also be used in the context of the present invention, the followingoligopeptides may be proposed in particular:

SerArgSerArgTyrTyrArgGlnArgGlnArgSerArgArgArgArgArgArg (COOH). (SEQ IDNo.7) and

ArgArgArgLeuHisArgIleHisArgArgGlnHisArgSerCysArgArgArgLysArgArg (COOH).(SEQ ID No. 8)

In the sense of the present invention, the term derivative denotes anypeptide, pseudopeptide (peptide incorporating non-biochemical elements)or protein differing from the protein or peptide considered, obtained byone or more genetic and/or chemical modifications. The expressiongenetic and/or chemical modification may be understood to mean anymutation, substitution, deletion, addition and/or modification of one ormore residues of the protein considered. More precisely, the termchemical modification refers to any modification of the peptide orprotein generated by chemical reaction or by chemical grafting ofbiological or non-biological molecule(s) onto any number of residues ofthe protein. The expression genetic modification is understood to referto any peptide sequence the DNA of which hybridizes with these sequencesor fragments thereof and the product of which possesses the activityindicated. Such derivatives may be generated for different purposes,such as in particular that of increasing the affinity of thecorresponding polypeptide or its (their) ligand(s), that of improvingits levels of production, that of increasing its resistance toproteases, that of increasing and/or modifying its activity, or that ofimparting novel pharmacokinetic and/or biological properties thereto.Among the derivatives resulting from an addition, mention may be made,for example, of chimeric peptide sequences containing a supplementaryheterologous part attached to one end. The term derivative alsocomprises protein sequences which are homologous with the sequenceconsidered, derived from other cell sources and in particular from cellsof human origin, or from other organisms, and possessing an activity ofthe same type. Such homologous sequences may be obtained byhybridization experiments on the corresponding DNA. The hybridizationsmay be performed with nucleic acid banks, using the native sequence or afragment thereof as probe, under conditions of conventional stringency(Maniatis et al.), (cf. General techniques of molecular biology), or,preferably, under conditions of high stringency.

In one particularly advantageous embodiment, the compositions of thepresent invention also comprise a targeting element which makes itpossible to direct the transfer of the nucleic acid. This targetingelement may be an extracellular targeting element, which allows thenucleic acid transfer to be directed towards certain types of cells orcertain desired tissues (tumour cells, liver cells, haematopoieticcells, and the like). Such an element may also be an intracellulartargeting element, allowing the nucleic acid transfer to be directedtowards certain favoured cell compartments (mitochondria, nucleus, andthe like).

The targeting element is more preferably linked, covalently ornon-covalently, to the compound according to the invention. Thetargeting element may also be linked to the nucleic acid. According to apreferred mode of the invention, the said compound is associated, via anadditional heterologous part bound to one of its ends. Such parts maybe, in particular, peptides of fusogenic type for promoting cellulartransfection, that is to say for favouring the passage of thetransfecting composition or its various elements across membranes, forhelping in the egress from endosomes or for crossing the nuclearmembrane. It may also be a cell receptor ligand present at the surfaceof the cell type, such as, for example, a sugar, transferrin, insulin orasialo-orosomucoid protein. Such a ligand may also be one ofintracellular type, such as a nuclear location signal (nls) sequencewhich promotes the accumulation of transfected DNA within the nucleus.

Among the targeting elements which may be used within the context of theinvention, mention may be made of sugars, peptides, hormones, vitamins,cytokines, oligonucleotides, lipids or sequences or fractions derivedfrom these elements and which allow specific binding with thecorresponding receptors. They are preferably sugars and/or peptides suchas antibodies or antibody fragments, cell receptor ligands or fragmentsthereof, receptors or receptor fragments, and the like. In particular,they may be ligands of growth factor receptors, of cytokine receptors,of cell lectin receptors or of adhesion protein receptors. Mention mayalso be made of the receptor for transferrin, for HDLs and for LDLs. Thetargeting element may also be a sugar which makes it possible to targetlectins such as the asialoglycoprotein receptors, or alternatively anantibody Fab fragment which makes it possible to target the Fc fragmentreceptor of immunoglobulins.

As an illustration of this type of association, use may be made inparticular, in the context of the present invention, of a compound oftype H₁-nls and more preferably a peptide possessing the sequenceProLysLysLysArgLysVal-βAlaLysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro (COOH) (SEQ IDNo.9).

Advantageously, the compound according to the invention and moreparticularly any histone derivative, protamine derivative or nucleolinederivative may also be polyglycosylated, sulphonated and/orphosphorylated and/or grafted to complex sugars or to a lipophiliccompound such as, for example, a polycarbon chain or a cholesterolderivative.

The composition according to the invention may obviously compriseseveral nucleic acid-compacting compounds, of different nature. It isthus possible to combine a compound of histone type with a compound ofnucleoline type.

The compound according to the invention is present in a sufficientamount to compact the nucleic acid according to the invention. Thus, thecompound/nucleic acid ratio (expressed in weight) may be between 0.1 and10 and more preferably between 0.3 and 3.

As regards the transfection agent present in the composition accordingto the invention, it is preferably chosen from cationic polymers andlipofectants.

According to the present invention, the cationic polymer is preferably acompound of general formula I,

in which

R may be a hydrogen atom or a group of formula

n is an integer between 2 and 10;

p and q are integers,

it being understood that the sum p+q is such that the average molecularweight of the polymer is between 100 and 10⁷ Da.

It is understood that, in the formula (I), the value of n may vary forthe different units p. Thus, the formula (I) combines both homopolymersand heteropolymers.

More preferably, in the formula (I), n is between 2 and 5. Inparticular, the polyethyleneimine (PEI) polymers and polypropylene4mine(PPI) polymers exhibit entirely advantageous properties. The polymerspreferred for carrying out the present invention are those whosemolecular weight is between 10³ and 5·10^(6.) By way of example, mentionmay be made of polyethyleneimine of average molecular weight 50,000 Da(PEISOK) or polyethyleneimine of average molecular weight 800,000 Da(PEI800K).

PRISOK and PEI800K are commercially available. As regards the otherpolymers represented by the general formula I, they may be preparedaccording to the process described in patent application FR 94/08735.

In order to obtain an optimum effect for the compositions of theinvention, the respective proportions of the polymer and the nucleicacid are preferably determined such that the molar ratio R=amines in thepolymer/phosphates in the nucleic acid is between 0.5 and 50, morepreferably between 5 and 30. Results which are most particularlyadvantageous are obtained using from 5 to 25 equivalents of polymeramines per charge of nucleic acid.

As regards more particularly the lipofectants, for the purposes of theinvention, any compound with a lipid nature and which has already beenproposed as an active agent with regard to the cellular transfection ofnucleic acids is understood to be covered by this name. In general,these are amphiphilic molecules comprising at least one lipophilicregion which may or may not be associated with a hydrophilic region.Representatives of the first family of compounds which may be proposedin particular are lipids capable of forming liposomes, such as POPCB,phosphatidylserine, phosphatidylcholine, cholesterol,maleimidophenylbutyrylphosphatidylethanolamine, lactosylceramide in thepresence or absence of polyethylene glycol to form furtive liposomes,or, with or without antibodies or ligands, to form immunoliposomes ortarget liposomes.

According to a particular mode of the invention, the lipid agent usedpossesses a cationic region. This cationically charged cationic region,preferably polyamine, is capable of combining reversibly with thenegatively charged nucleic acid. This interaction strongly compacts thenucleic acid. The lipophilic region renders this ionic interactioninaccessible to the external aqueous medium, by coating the. nucleolipidparticle formed with a lipid film.

Thus, it is known that a positively charged cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA),interferes, in the form of liposomes or small vesicules, spontaneouslywith DNA, which is negatively charged, to form lipid-DNA complexescapable of fusing with cell membranes, and thereby allows the DNA to bedelivered into the cell. Since DOTMA, other cationic lipids have beenproposed along this structural model: lipophilic group associated withan amino group via a so-called “spacer” arm. Among these, mention may bemade more particularly of those comprising, as lipophilic group, twofatty acids or a cholesterol derivative, and also containing, whereappropriate, as amino group, a quaternary ammonium group. DOTAP, DOBTand ChOTB may be mentioned in particular as representatives of thiscategory of cationic lipids. Other compounds, such as DOSC and ChOSC,are characterized by the presence of a choline group in place of thequaternary ammonium group. Another category of cationic lipids,lipopolyamines, has also been described. In compounds of this type, thecationic group is represented by the L-5-carboxyspermine radical whichcontains four ammonium groups, two primary and two secondary. DOGS andDPPES are particularly among these lipopolyamines. These lipopolyaminesare most particularly effective- for transfecting primary endocrinecells.

Advantageously, the lipofectants suited to the invention may also bechosen from lipopolyamines whose polyamine region corresponds to thegeneral formula (II)

in which m is an integer greater than or equal to 1 and n is an integergreater than or equal to 1, it being possible for m to vary for thedifferent carbon groups between two amines. Preferably, m is between 2and 6 inclusive and n is between 1 and 5 inclusive. Even morepreferably, the polyamine region is represented by spermine, thermine orone of the analogues thereof which has conserved the properties ofbinding to the DNA. As regards the lipophilic region, it is representedby at least one saturated or unsaturated hydrocarbon chain, cholesterol,a natural lipid or a synthetic lipid capable of forming lamellar orhexagonal phases, linked covalently to the hydrophilic region.

Patent application EP 394,111 describes other lipopolyamines of generalformula III which may be used within the context of the presentinvention:

in which R represents in particular a radical of general formula(R₁R₂)N—CO—CH—NH—CO—.

Representative examples of these lipopolyamines which may be mentionedmore particularly are dioctadecylamidoglycylspermine (DOGS) andpalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES).

The lipopolyamines described in patent application FR 94/14596 may alsobe used advantageously as transfection agent according to the invention.They are represented by the general formula IV above in which Rrepresents

with

X and X′ representing, independently of each other, an oxygen atom, amethylene group —(CH₂)_(q)— with q equal to 0, 1, 2 or 3, or an aminogroup

—NH— or —NR′— with R′ representing a C₁ to C₄ alkyl group,

Y and Y′ representing, independently of each other, a methylene group, acarbonyl group or a C=S group,

R₃, R₄ and R₅ representing, independently of each other, a hydrogen atomor a substituted or unsubstituted C₁ to C₄ alkyl radical, with it beingpossible for p to range between 0 and 5,

R₆ representing a cholesterol derivative or an alkylamino group —NR₁R₂with R₁ and R₂ representing, independently of each other, a saturated orunsaturated linear or branched C₁₂ to C₂₂ aliphatic radical.

Representative examples of these lipopolyamines which may be mentionedmost particularly are 2,5-bis(3-aminopropylamino)pentyl(dioctadecylcarbamoylmethoxy)acetate and1,3-bis(3-aminopropylamino)-2-propyl(dioctadecylcarbamoylmethoxy)acetate, hereinafter referred to aslipopolyamine A.

Patent applications EP 394,111 and FR 94 145 96 also describe a processwhich may be used for the preparation of the correspondinglipopolyamines.

Lastly, more recently, novel lipopolyamines, which can also be upgradedwithin the context of the present invention, have been described inpatent application FR 95/134 90. Representatives of these lipopolyamineswhich may be mentioned more particularly are the following:

lipopolyamine B:

{H₂N(CH₂)₃}₂N(C₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGln[(CH₂)₁₇—CH₃]₂RP120525)

lipopolyamine C:

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₈]₂ (RP120535)

lipopolyamine D:

H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂CoArggN[(CH₂)₁₈] (RP120531)

Within the context of the invention, dioctadecylamidoglycylspormine(DOGS), palmditoylphosphatidylethanolamine 5-carboxyspermylamide(DPPES), 2,5-bis(3-aminopropylamino)pentyl(dioctadecylcarbamoylmethoxy)acetate,1,3-bis(3-aminopropylamino)-2-propyl(dioctadecylcarbamoylmethoxy)acetate,

{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[((CH₂)₁₇—CH₃]₂,H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₈]₂ or

H₂N(CH₂)₃NH (CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₈] may be used particularlyadvantageously.

In order to obtain an optimum effect for the compositions of theinvention, the respective proportions of the polyamine and the nucleicacid are preferably determined such that the ratio R of positive chargeson the transfection agent/negative charges on the nucleic acid isbetween 0.1 and 10 and more preferably between 0.5 and 6.

The presence of a compound according to the invention within atransfecting composition makes it advantageously possible to decreasethe amount of transfection agent considerably. This results in amarkedly reduced toxicity which consequently makes possible, forexample, the transfection of cells which are sensitive at the source tothe transfection agent such as, for example, haematopoietic cells withthe lipopolyamines. Lastly, as the examples below demonstrate, thetransfecting power of the compositions according to the invention isgreater than that obtained with the standard transfecting compositions.

In the compositions of the present invention, the nucleic acid mayeither be a deoxyribonucleic acid or a ribonucleic acid. It may besequences of natural or artificial origin, and in particular genomicDNA, cDNA, mRNA, tRNA, rRNA, hybrid sequences or synthetic orsemi-synthetic sequences. These nucleic acids may be of human, animal,plant, bacterial, viral, etc. origin. They may be obtained by anytechnique known to those skilled in the art, and in particular by thescreening of banks, by chemical synthesis or by mixed methods includingthe chemical or enzymatic modification of sequences obtained by thescreening of banks. They may moreover be incorporated into vectors, suchas plasmid vectors.

As regards more particularly the deoxyribonucleic acids they may besingle- or double-stranded. These deoxyribonucleic acids may code fortherapeutic genes, sequences for regulating transcription orreplication, antisense sequences, regions for binding to other cellcomponents, etc.

In the sense of the invention, the term therapeutic gene is understoodin particular to refer to any gene which codes for a protein producthaving a therapeutic effect. The protein product thus encoded may be aprotein, a peptide, etc. This protein product may be homologous withrespect to the target cell (that is to say a product which is normallyexpressed in the target cell when the latter exhibits no pathology). Inthis case, the expression of a protein makes it possible, for example,to overcome an insufficient expression in the cell or the expression ofa protein which is inactive or weakly active on account of amodification, or alternatively of overexpressing the said protein. Thetherapeutic gene may thus code for a mutant of a cell protein, havingincreased stability, modified activity, etc. The protein product mayalso be heterologous with respect to the target cell. In this case, anexpressed protein may, for example, make up or provide an activity whichis deficient in the cell, enabling it to combat a pathology or tostimulate an immune response.

In the sense of the present invention, among the therapeutic productswhich may more particularly be mentioned are enzymes, blood derivatives,hormones, lymphokines, interleukins, interferons, TNF, etc. (FPR9203120), growth factors, neurotransmitters or their precursors orsynthetic enzymes, trophic factors: BDNF, CNTF, NGF., IGF, GMF, aFGF,bFGP, VEGF, NT3, NT5, HARP/pleiotrophin, genes corresponding to theproteins involved in the metabolism of lipids, of apolipoprotein typechosen from apolipoproteins A-I, A-II, A-IV, B, C-I, C-II, C-III, D, E,F, G. H, J and apo (a), metabolic enzymes such as, for example,lipoprotein lipase, hepatic lipase, lecithin cholesterolacyltransferase, 7-alpha-cholesterol hydroxylase, phosphatidic acidphosphatase, or lipid transfer proteins such as cholesterol estertransfer protein and phospholipid transfer protein, a protein forbinding HDLs or a receptor chosen, for example, from LDL receptors,chylomicron-remnant receptors and scavenger receptors, dystrophin orminidystrophin (FR 9111947), GAX protein, CFTR protein associated withmucoviacidosis, tumour-suppressant genes: p53, Rb, Rap1A, DCC, k-rev,etc. (FR 9304745), genes coding for factors involved in coagulation:factors VII, VIII, IX, genes involved in DNA repair, suicide genes(thymidine kinase, cytosine deaminase), etc.

The therapeutic gene may also be an antisense sequence or a gene whoseexpression in the target cell makes it possible to control theexpression of genes or the transcription of cellular NA. Such sequencesmay, for example, be transcribed in the target cell into complementaryRNA of cellular mRNA and thus block their translation into protein,according to the technique described in patent EP 140,308. The antisensesequences also comprise the sequences coding for ribozymes which arecapable of selectively destroying target RNA (EP 321,201).

As indicated above, the nucleic acid may also contain one or more genescoding for an antigenic peptide, capable of generating an immuneresponse in humans or animals. In this particular embodiment, theinvention thus makes it possible to produce either vaccines orimmunotherapeutic treatments applied to humans or to animals, inparticular against microorganisms, viruses or cancers. They may inparticular be antigenic peptides specific for Epstein Barr virus, forHIV virus, for hepatitis B virus (EP 185,573), for pseudo-rabies virusor alternatively specific for tumours (EP 259,212).

Preferably, the nucleic acid also comprises sequences which allow theexpression of the therapeutic gene and/or of the gene coding for theantigenic peptide in the desired cell or organ. These may be sequenceswhich are naturally responsible for expression of the gene consideredwhen these sequences are capable of functioning in the infected cell.They may also be sequences of different origin (responsible for theexpression of other proteins, or even synthetic). In particular, theymay be promoter sequences for eukaryotic or viral genes. For example,they may be promoter sequences derived from the genome of the cell whichit is desired to infect. Similarly, they may be promoter sequencesderived from the genome of a virus. In this regard, there may forexample be mentioned the promoters of genes E1A, MLP, CKV, RSV, etc. Inaddition, these expression sequences may be modified by addition ofactivation sequences, regulation sequences, etc.

Moreover, the nucleic acid may also contain, in particular upstream ofthe therapeutic gene, a signal sequence which directs the therapeuticproduct synthesized into the secretion pathways of the target cell. Thissignal sequence may be the natural signal sequence of the therapeuticproduct, but it may also be any other functional signal sequence, or anartificial signal sequence.

More preferably, the compositions of the invention also comprise one ormore neutral lipids. Such compositions are particularly advantageous,especially when the ratio R is low. The Applicant has indeed shown thatthe addition of a neutral lipid makes it possible to improve theformation of the nucleolipid particles and, surprisingly, to promote thepenetration of the particle into the cell by destabilizing its membrane.

More preferably, the neutral lipids used in the context of the presentinvention are lipids containing 2 fatty chains.

In a particularly advantageous manner, natural or synthetic lipids,which may be zwitterionic or devoid of ionic charge under thephysiological conditions, are used. They may be chosen more particularlyfrom dioleoylphosphatidylethanolamine (DOPE),oleoylpalmitoylphosphatidylethanolamine (POPE), di-atearoyl, -palmitoylor myristoyl phosphatidylethanolamine as well as derivatives thereofN-methylated 1 to 3 times, phosphatidylglycerols, diacylglycerols,glycosyldiacylglycerols, cerebrosides (such as galactocerebrosides inparticular), sphingolipids (such as sphingomyelins in particular) oralternatively asialogangliosides (such as asialoGM1 and GM2 inparticular).

These various lipids may be obtained either by synthesis or byextraction from organs (example: brain) or from eggs, by standardtechniques well known to those skilled in the art. In particular, theextraction of natural lipids may be performed using organic solvents(see also Lehninger, Biochemistry).

The compositions of the invention, using a lipofectant as transfectionagent, preferably comprise from 0.1 to 20 equivalents of neutral lipidper one equivalent of lipopolyamine and, more preferably, from 1 to 5.In the case in which the transfection agent is a cationic polymer, thecompositions of the invention comprise, in addition to the cationicpolymer in the ratios mentioned above, from 0.1 to 20 molar equivalentsof neutral lipid per 1 molar equivalent of nucleic acid phosphate, andmore preferably from 1 to 5.

The compositions according to the invention may be formulated for thepurpose of topical, cutaneous, oral, rectal, vaginal, parenteral,intranasal, intravenous, intramuscular, subcutaneous, intraocular,transdermal, etc. administration. The pharmaceutical compositions of theinvention preferably contain a vehicle which is pharmaceuticallyacceptable for an injectable formulation, in particular for directinjection into the desired organ, or for topical administration (to skinand/or mucous membrane). They may in particular be sterile, isotonicsolutions or dry compositions, in particular freeze-dried compositions,which, by addition, depending on the case, of sterilized water or ofphysiological saline, allow injectable solutions to be made up. Thedoses of nucleic acid used for the injection and the number ofadministrations may be adapted according to various parameters, and inparticular according to the mode of administration used, the pathologyconcerned, the gene to be expressed, or alternatively the desiredduration of the treatment.

They may advantageously be used to transfect a wide variety of celltypes such as, for example, haematopoietic cells, lymphocytes,hepatocytes, endothelial cells, melanoma carcinoma and sarcoma cells,smooth muscle cells, neurons and astrocytes.

The present invention thus provides a particularly advantageous methodfor the treatment of diseases, using the in vivo, ex vivo or in vitrotransfection of a nucleic acid capable of correcting the said disease,in combination with a transfection agent of cationic polymer orlipofectant type, and a compound as defined above. More particularly,this method may be applied to diseases resulting from a deficiency of aprotein or nucleic acid product and the nucleic acid administered codesfor the said protein product or contains the sequence corresponding tothe said nucleic acid product. The compositions according to theinvention are particularly advantageous on account of theirbioavailability and their high level of transfection.

The present invention also relates to any use of a compound consisting,partly or totally, of peptide units (LysThrProLysLysAlaLysLysPro) (SEQID No.: 1) and/or (AlaThrProAlaLysLysAlaAla) (SEQ ID No.: 2) with itbeing possible for the number of these units to range between 2 and 10,in order, when they are coupled to a cell receptor ligand, an antibodyor an antibody derivative, to target a nucleic acid towards cells whichexpress the corresponding receptors or antigens. In this perspective, apotential ligand, antibody or antibody derivative is coupled to the saidcompound and the transfection power of this chimeric molecule isassessed relative to the compound alone.

The present invention also covers any use of an oligopeptide selectedfrom:

(AlaThrProAlaLysLysAlaAlaAlaThrProAlaLysLysAlaAla) (COOH) (SEQ ID No.:3), (LysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro) (COOH) (SEQID No.: 4), AlaThrProLysLysSerAlaLysLysThrProLysLysAlaLysLysPro (COOH)(SEQ ID No.: 5),LysLysAlaLysSerProLysLysAlaLysAlaAlaLysProLysLysAlaProLysSerProAlaLysAlaLysAla (COOH) (SEQ ID No.: 6),SerArgSerArgTyrTyrArgGlnArgGlnArgSer-ArgArgArgArgArgArg (COOH) (SEQ IDNo.: 7) andArgArgArgLeuHisArgIleHisArgArgGlnHisArgSerCysArgArgArgLysArgArg (COOH)(SEQ ID No. 8)

in order to carry out the in vitro, ex vivo and/or in vivo transfer ofat least one nucleic acid, the said oligonucleo- tide being or not beingassociated with a targeting element.

The present invention will be described more fully using the exampleswhich follow, which should be considered as being non-limitingillustrations.

Equipment and methods:

Plasmids used for the in vivo transfer of genes

The constructions used to demonstrate the activity of the compositionsof the invention are plasmids containing the gene coding for luciferase(Luc).

These plasmids are, in particular, pCXV luc, pXL 2621 and pXL 2622,which all contain the gene coding for luciferase (taken from the vectorpGL2, Promega) downstream of the cytomegalovirus (CMV) promoterextracted from pCDNA3 (Invitrogen). pCMV luc and pXL 2622 are derivedfrom a pGL2 vector, pXL 2621 from a control pGL2 vector, and, in allthese vectors, the SV40 promoter has been replaced by the CMV promoter.

In general, the plasmids are obtained by the technique of precipitationwith PEG (Ausubel) and stored in 10 mM tris 1 mM EDTA pH 8 at 4° C. at aconcentration of about 10 μg of DNA per μl.

H: LysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro (COOH) (SEQ IDNo.: 4) 18AA.

N: AlaThrProAlaLysLysAlaAlaAlaThrProAlaLysLysAlaAla (COOH) (SEQ ID No.:3) 16AA

nls-H:ProLysLysLysArgLysValβAlaLysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro(COOH) (SEQ ID No.: 9) 26AA;

PR1:

ArgArgArgLeuHisArgIleHisArgArgGlnHisArgSerCysArg ArgArg-LysArgArg (SEQID No.: 8) 21AA

PR2:

SerArgSerArgTyrTyrArgGlnArgGlnArgSerArgArgArgArgArgArg (SEQ ID No.: 7)

They were prepared as follows:

2.1 N: AlaThrProAlaLysLysAlaAlaAlaThrProAlaLysLysAlaAla (COOH) (SEQ IDNo.: 3)

This oligopeptide was synthesized in the form of the trifluoroaceticacid salt using an Applied Biosystem 431A peptide synthesizer, on an HMPresin (Applied Biosystem) and according to an FMOC strategy. After thesynthesis, the peptide was released from the resin by treatment for 90minutes in the presence of {fraction (1/19)} water/TFA solution. Afterfiltration, the solution is concentrated on a rotary evaporator and thepeptide is then precipitated twice, by addition of tert-butyl methylether, from a solution in TFA. The final pellet is washed withtert-butyl methyl ether and then dried. The peptide is dissolved in 5 mlof water, filtered and purified by reverse-phase HPLC on a C18 100 Acolumn (Biorad RSL). The peptide is purified by with the aid of agradient of 0 to 25% of acetonitrile, 0.07% TFA in water 0.07% TFA. Thepurity of the peptide obtained is greater than 95% and its solubility inwater is 100 mg/ml.

2.2 nls: ProLysLysLysArgLysVal (SEQ ID. NO: 9, residues 1-7)

This oligopeptide was synthesized in the form of the trifluoroaceticacid salt according to the procedure described above, using 2/40/3/1/2(v/v) water/TFA/phenol/ethanedithiol/thioanisole solution for thecleavage. The purity of the peptide obtained is greater than 95% and itssolubility in water is 100 mg/ml at pH 2.1.

2.3 H: LysThrProLysLysAlaLysLysProLysThrProLysLysAlaLysLysPro (COOH)(SEQ ID No.: 4) and nls-H:ProLysLysLysArgLysVal-β-AlaLysThrProLysLysAlaLysLysProLysThrProLysLvsAlaLysLysPro(COOH)(SEQ ID No.:9)

These oligopeptides are synthesized in the form of the trifluoroaceticacid salts according to the procedure described above. To do this, theresin is divided into two batches. A first batch intended for thesynthesis of H is treated for the cleavage with 40/3/1/2/2 (v/v)TFA/phenol/ethanedithiol/thioanisole/water solution. The purity of thepeptide obtained is greater than 90% and its solubility in water is 10mg/ml at pH 2.1. The synthesis is continued on the second batch of resinso as to obtain nls-β-Ala-H. The cleavage solution used is identical tothe previous one. The purity of the peptide obtained is greater than 95%and its solubility in water is 10 mg/ml at pH 2.1.

2.4 PR1: ArgArgArgLeuHisArgtIleHisArgArgIleHisArgSerCysArgArgArgLysArgArg (SEQ ID No.: 8) and PR2:SerArgSerArgTyrTyrArgGlnArglnArgaerArgArgArgArgArgArg (SEQ ID No.: 7)

These oligopeptides are assembled in several steps, in a solid phasesynthesis according to the BocBenzyl technique. The starting resin is aBoc-L-Arg(Tos)Pam resin (0.48 meq/g). The deprotection and couplingprocess used is as follows:

1- 55% TFA in dichloromethane (DCM) 1 × 2 mn 2- 55% TFA indichloromethane 1 × 30 mn 3- DCM 2 × 1 mn 4- DMF 3 × 1 mn 5- Coupling 6-DMF 2 × 1 mn 7- DCM 2 × 1 mn

For each step, 10 ml of solvent are used per gram of peptide resin used.The coupling of all the amino acids (in three-fold excess) is carriedout in DMF in the presence of BOP, Hobt and DIEA. Each coupling step iscontrolled by the ninhydrin test.

The final peptide is recovered from the resin and deprotected fully withliquid hydrofluoric acid. 10 ml of HF are used per gram of resin peptideat 0° C. for 45 minutes in the presence of para-cresol andethanedithiol. After evaporation of the hydrofluoric acid, the crudereaction mixture is washed with ether, dissolved in trifluoroaceticacid, precipitated with ether and dried.

3. Lilofectant agents used

Lipopolyamine A:

d,1-3-bis(3-aminopropylamino)-2-propyl(dioctadecylcarbamoylmethoxy)acetate (RP115335)

Lipopolyamine B:

{H₂N(CH₂)₃}₂N(CH₂)₄N{(C₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂ (RP120525)

Lipopolyamine C:

H₂N (CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₈]₂(RP120535)

Lipopolyamine D:

H₂N (CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₈]₂ (RP120531)

EXAMPLE 1

in vitro transfer of nucleic acid into NIH 3T3 cells

This example describes the in vitro transfer of nucleic acids (on cellcultures) using a composition according to the invention comprising thenucleic acid, a compound according to the invention and a lipopolyamine,under various pH and buffer conditions.

1. A mixture of 10 μl composed of the following is prepared:

0.5 μg of pCKV-luc plasmid DNA, 0.25 μg of a compound according to theinvention, in a buffer solution as identified,

40 mM dioctadecylamidoglycylspermine (DOGS) in charge ratios X asindicated in each of the tests.

2. 5·10⁴ cells from the NIH3T3 lines are incubated with the abovemixture at 37° C., under an atmosphere of 5% CO₂ for 4 hours. The cellsare then washed and recultured for 48 hours in a medium containing 10%serum (DMEM 10% SVF).

The cell carpet is then lysed in 50 μl of lysis buffer (Promega),recovered and then centrifuged at 20,000 g for 5 minutes.

The luciferase activity is measured on 4 μl of supernatant by adding 20μl of substrate (Promega). The reading is taken on a LKB luminometer,cumulating the RLUs (relative light units) over 20 seconds.

The results are given in Tables 1 and 2 below.

3. Compaction in 150 mM NaCl, 10 MM HEPES* (*N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulphonic acid) at pH 7.1

TABLE 1 DOGS/DNA charge Without compound With H ratio (R.L.U) (R.L.U)0.8×  32  850 1.8×  220 23700   3× 5400 21750

4. Compaction in 5% D-glucose. 150 mM NaCl, with 10 mM HEPES (pH of thecompaction solution: 7.2)

TABLE 2 Without DOGS/DNA compound With H With N charge ratio (R.L.U)(R.L.U) (R.L.U) 0.8×  880  44000  1450 1.8×  6600 156500 —   3× 22000297500 90300

Better results are observed in all of the tests when the compactingcomposition combines a compound according to the invention with DOGS. Itturns out to be possible to decrease substantially the amount of DOGSwithout having a prejudicial effect on the transfection capacity of thecorresponding composition.

EXAMPLE 2

Transfection test in the presence of a neutral livid

A mixture of 10 μl composed of the following is prepared:

0.5 μg of pCMV-luc plasmid DNA, 0.25 μg of a compound according to theinvention, in 150 mM NaCl buffer and 10 μM phosphate buffer solution, pH7.4,

40 mM of dioctadecylamidoglycylspermine (DOGS) in charge ration X asindicated in each of the tests, in the presence ofdioleoylphosphatidylethanolamine (DOPE) with DOGS/DOPE equal to ½.

In this particular case, a 40 mM ethanolic DOGS solution is mixed withan equal volume of an 80 mM dioleoylphosphatidylethanolamine (DOPE)solution, prepared in a chloroform/ethanol (⅕) mixture. Thus, for oneequivalent of DOGS the composition contains two equivalents of DOPE.

10⁵ cells from NIH3T3 lines are incubated with this mixture under theconditions described in the above example. After incubation, these cellsare treated according to the procedure of the same example. The resultsare given in the table below.

TABLE 3 Without With nls-H DOGS/DNA compound With H chimera charge ratio(RLU) (RLU) (RLU) 0.8 × D/D 24 3175  760   1 × D/D 19 7410  2380 1.8 ×D/D 1800  39500  52800

These results confirm those observed in Example 1. In the presence of abasic compound according to the invention, and more particularly H, itis possible to reduce the amount of lipofectant by half.

EXAMPLE 3

Variation of the compound according to the invention/DNA ratio within atransfecting composition according to the invention.

3.1: In the presence of a DOGS composition

1. A mixture of 10 μl composed of the following is prepared:

0.75 μg of pCMV-luc plasmid DNA and a compound according to theinvention in the ratio indicated, in 5% D-glucose, 150 mM NaCl buffersolution, with 10 mM HEPES (pH of the compaction solution: 7.2),

40 mM dioctadecylamidoglycylspermine (DOGS) in a charge ratio of 1.8 X.

2. 4·10⁵ cells from NIH3T3 lines are incubated with the above mixture at37° C., under an atmosphere of 5% CO₂ for 4 hours. The cells are thenwashed and recultured for 48 hours in a medium containing 10% serum(DMEM 10%SVF). The cell carpet is then lysed, 45 hours after thetransfection, in 100 μl of lysis buffer (Promega), recovered and thencentrifuged at 20,000 g for 5 minutes. The luciferase activity ismeasured on 5 μl of the supernatant, adding 25 μl of substrate(Promega). The reading is taken on an LKB luminometer, cumulating theRLUs (relative light units) over 20 seconds.

The results are featured in Table 4 below.

TABLE 4 PEP/DNA With H With H-nls With N w/v (RLU) (RLU) (RLU) 0 21502150 2150 0.25 3300  38,000 35,000 0.5 45,000 100,000 35,000 1 84,000105,000 13,000 2 105,000  200,000 20,000

3.2: In the presence of 1.8X DOGS/DOPE

The process is performed as in the procedure described above in 3.1, butusing as transfecting agent a 40 mM solution of 1.8×dioctadecylamidoglycylspermine (DOGS) in the presence ofdioleoylphosphatidyl-ethanolamine (DOPE) with DOGS/DOPE equal to ½,prepared according to the procedure presented in Example 2.

Table 5 gives the results observed.

TABLE 5 COMPOUND/DNA H H-nls N w/v (RLU) (RLU) (RLU) 0 580  580  5800.25 13,000 5600 7600 0.5 11,000 18,500 1800 1 14,100 36,000 13,600 216,500 58,000 14,100

EXAMPLE 4

Variation of the compound according to the invention/DNA ratio within atransfecting composition according to the invention, using atransfection agent other than DOGS.

3.1: In the Presence of 1.3-bis-(3-aminopropylamino)-2-propyl(dioctadecylcarbamoylmethoxy)acetate (lipopolyamine A)

1. A mixture of 10 μl composed of the following is prepared:

0.55 μg of pCMV-luc plasmid DNA and a compound according to theinvention, in the ratio indicated, in 150 μm NaCl buffer solution,

1,3-bis (3-aminopropylamino)-2-propyl(dioctadecylcarbamoylmethoxy)acetate in in the charge ratios indicated.

2. 5-10⁴ cells from NIH3T3 lines are incubated with the above mixture at37° C., under an atmosphere of 5% CO₂ for 4 hours. The cells are thenwashed and recultured for 48 hours in a medium containing 10% serum(DMEM 10%SVF). The cell carpet is then lysed, 45 hours after thetransfection, in 100 μl of lysis buffer (Promega), recovered and thencentrifuged at 20,000 g for 5 minutes. The luciferase activity ismeasured on 10 μl of the supernatant, adding 50 μl of substrate(Promega). The reading is taken on a Berthold lumat 950® luminometer,cumulating the RLUs (relative light units) over 10 seconds. The resultsare featured in Table 6 below.

TABLE 6 NH₂ in the LIPOFECTANT lipofectant/ Without phosphates compound· With With in the 10⁶ · RLU H · 10⁶ · RLU nls-H · 10⁶ · RLU DNA ratioCompound/DNA ratio 0 0.5 1 2 0.5 1 2 1.8× 3.9 30.4 32.7 4.1 52.4 58.539.3   3× 8.9 41.5 61.3 16.6 64.1 60.4 49.2   6× 6.2 23.6 22.3 15.6

4.2: In the presence of PEI 800K

1. The process is performed according to the procedure described in 4.1and in an identical medium, using PEI800K as lipofectant. The resultsare featured in Table 7 below.

TABLE 7 Equivalent PEI 800K of amines Without in the polymer compound ·to phosphate 10⁶ · RLU H · 10⁶ · RLU nls-H · 10⁶ · RLU in the DNACompound/DNA ratio 0 0.5 1 2 0.5 1 2 6 7.7 4.9 7 12.3 4 7.3 8.8 9 5 811.6 4 16.3 11 12.1 12  7.5 11.3 17.1 1.2

EXAMPLE 5

In vivo transfer into muscle cells

The corresponding tests are performed using the following procedures andmaterials:

Model: The injection is made into the cranial tibial muscle of adult C57b16 or OP1 mice (more than 8 weeks old)

Procedure: The DNA is diluted to 0.5 mg/ml in a solution which will havefinal concentrations of 150 nM NaCl and 5% D-glucose. In certain groups,before injection, peptide as a 1 mg/ml solution in water is added to theDNA in an amount which is sufficient to reach the weight/weight ratiosindicated. Incubation for at least 20 minutes at room temperature iscarried out before the injection is made. Determination of the results:Two days after the injection, the muscle is removed and chopped up in750 μl of lysis buffer (Promega E153A) supplemented with aprotinin(Sigma).

The sample is homogenized in a grinder (Heidolf) and 10 μl are used tomeasure the luciferase activity. This measurement is taken with a Lumat9501 luminometer (Berthold), by totalizing the emission produced for 10seconds after addition of 50 μl of luciferase substrate (Promega) to 10μl of the sample. The background noise measured before addition ofsubstrate is subtracted from this total, and the activity is expressedas total RLU (relative light units) (relative to 750 ml of lysisbuffer).

To do this, 40 μl (20 μg of DNA) are injected, in the presence of HEPESat pH 7.4 final concentration of 5 mM in the solution, followed byaddition of the lipopolyamine C(RPR 120 535) in a ratio 0.01 nmol/μl ofDNA 20 minutes before injection:

TABLE 8 Peptide, RLU number peptide/DNA Average standard ofweight/weight ratio RLU deviation animals without peptide 15 228 125 14618 681 6 nls-H, 0.025 42 722 500 66 485 348 6 weight/weight +lipopolyamine C

These results confirm the beneficial effect of the combination of alipopolyamine with a compacting agent according to the invention on thein vivo transfection of a nucleic acid into muscle.

EXAMPLE 6

In vivo transfer of compositions claimed into tumour

cells

The corresponding tests are carried out on adult (>9 weeks) femaleC57/BL mice carrying tumours of type 3LL (Lewis Lung carcinoma) obtainedby passing fragments of tumour from animal to animal, implanted underthe skin of the flank.

As regards the solutions injected, they are prepared as follows: the DNAis first dissolved in the buffer, the peptide is then optionally addedand, after 20 minutes, a solution of cationic lipids at highconcentration (20 or 40 mM) is added to the mixture. After addition ofall these products, the mixture contains, besides the DNA, the peptideand the cationic lipid, 150 mM NaCl, 5% D-glucose and 5 mM MES pH 6.2.In the case of the last two series with lipopolyamine C (RPR 120 535),the injection vehicle is 75 mM NaCl and 150 =M NaCl, 5% D-glucoserespectively. The injection is made 20 to 30 minutes after the solutionhas been prepared.

Each transfecting composition (see Tables 9 and 10 for their respectivespecificities) is injected into the tumour 7 days after implantation,the mouse being anaesthetized with a ketamine 130 mg/kg+xylazine (4mg/kg) mixture.

Two days after the injection, the tumour tissue is removed, weighed andthen chopped up and ground in 500 μl of lysis buffer (Promega cell lysisbuffer E153 A). After centrifugation (20,000 g for 10 minutes), 10 μlare taken and used to evaluate the luciferase activity by measuring thetotal light emission obtained after mixing with SO ;l of reagent(Promega luciferase assay substrate) in a Lumat LB 9501 luminometer(Berthold), with integration over 10 seconds.

The resulting activity is expressed as RLUs (relative light units)estimated in the entire tumour lysis supernatant, or as RLU per μg ofDNA injected.

Table 9 gives the results obtained in the presence of variouslipopolyamines A, B, C or D and Table 10 in the presence of PEI.

TABLE 9 Result, Number Plasmid Peptide Cationic lipid RLU/tumour of μg/[DNA] pept/DNA nmol/ standard animals tumour μg/μl reference w/wreference μg DNA average deviation treated 20 2 0 0 5 20 2 A 1.8 142 300121 418 5 20 2 H 1 1.8 301 730 243 166 5 30 2 3 99 775 128 726 6 30 2 H1 3 1 340 460 1 771 624 5 7.5 0.5 A 3 88 712 49 314 5 7.5 0.5 H 1 3 383313 234 713 6 7.5 0.5 H 1.5 3 618 025 530 774 6 15 1 H 1 3 1 017 372 966141 5 10 0.5 H 1.5 C 3 679 258 414 286 9 10 0.5 H 1.5 D 3 395 433 219333 10  18.75 0.25 C 2 222 700 126 036 6 18.75 0.25 Pr 2 0.5 ″ 2 1 046050 612 401 6 20 0.5 C 4 806 467 887 206 6 20 0.5 H 1 C 4 1 348 233 1674 106 6

TABLE 10 Result, Number Plasmid Peptide Poly- RLU/tumour of amount [DNA]peptide/ ethyleneimine standard animals injected μg/μl reference DNA w/wsize Eq average deviation treated 20 2 0 0 5 20 2 800K  9 54 350 52 9895 50 2 800K  9 7 783 16 803 6 50 2 H1 1 800K  9 62 230 71 462 5 50 2800K 12 6 733 16 493 6 50 2 H1 1 800K 12 72 700 150 300 5 40 2 800K 18470 1 051 5 40 2 H1 1 800K 18 82 608 104 443 6 40 2 800K 24 1 630 3 6455 40 2 H1 1 800K 24 45 750 63 942 5 10 0.5 H1 1.5  50K 12 14 152 16 94611  lactose 10 0.5 H1 1.5  50K 12 12 942 22 853 11  maltose

EXAMPLE 7

In vitro transfer of nucleic acid into 3LL cells

This example describes the in vitro transfer of nucleic acids (on cellcultures) using a composition according to the invention comprising thenucleic acid, a compound according to the invention chosen fromprotamine derivatives and a lipopolyamine in a solution of 75 mM NaClfinal.

A mixture of 10 μl composed of the following is prepared:

0.5 μg of pCMV-luc plasmid DNA, 0.5 μg of PR1 or PR2 a compoundaccording to the invention, in a solution of 75 mM NaCl final

lipopolyamine C (RPR 120535) in charge ratios as indicated in each ofthe tests listed in Table 11 below.

1×10⁵ ³LL cells (in 250 μl of DMEM culture medium with 10% foetal calfserum) are incubated with the above mixture at 37° C. under a 5% CO₂atmosphere for 4 hours. 500 μl of culture medium are added and the cellsare recultured. The following day, the cells are washed and reculturedfor 24 hours in the same medium containing 10% foetal calf serum.

The cell carpet is then lysed in 100 μl of lysis buffer (Promega). Theluciferase activity is measured by adding 50 μl of substrate (Promega).The reading is taken on an LB luminometer by cumulating the RLUs(relative light units) over 10 seconds.

Tables 11 and 12 give the tests with PR1 and PR2 respectively.

TABLE 11 Lipopolyamine C/DNA WITHOUT COMPOUND WITH PR2 CHARGE RATIO(RLU) (RLU) 4× 107 289  447 214 6× 77 396 1 182 641  8× 14 512 729 285

TABLE 12 Lipopolyamine C/DNA WITHOUT COMPOUND WITH PR2 CHARGE RATIO(RLU) (RLU) 4× 12 037   6 304 6× 901 113 113 8× 328 294 743

EXAMPLE 8

In vivo injections of compacting peptides according to the invention,without lipofectant, into tumours

Model:

mice of adult female nude/Swiss type

experimental tumours induced after injection of 10⁷3T3 HER2 cellssubcutaneously into the flank.

injection of the transfection mixture 7 to 12 days after injecting thecalls. The 'solution of DNA which is or is not compacted with peptide isinjected directly into the tumour with a Hamilton-type syringe.

two days after the injection, the tumour tissue is removed, weighed andthen chopped up and homogenized in 750 μl of lysis buffer (Promega celllysis buffer E153 A). After centrifugation (20,000 g for 10 minutes), 10μl are removed and are used to evaluate the luciferase activity bymeasuring the total light emission obtained after mixing with 50 μl ofreagent (Promega luciferase assay substrate) in a Lumat LB 9501luminometer (Berthold) with integration over 10 seconds.

The resulting activity is expressed as RLUs (relative light units)estimated in the entire tumour lysis supernatant.

Procedure: The DNA is diluted to 0.5 mg/ml in a solution which willcontain a final concentration of the salts, the buffer and the glucosein a final amount as stated in the table of results. In certain groups,before injection, peptide dissolved to 1 mg/ml in water is added to theDNA in an amount which is sufficient to reach the weight/weight ratiosindicated. Incubation for at least 20 minutes at room temperature iscarried out. The mice receive an injection of 20 μl, i.e. 10 μg of DNAin total per tumour.

TABLE 13 Buffer NaCl/ Peptide Result, Number glucose/ peptide/RLU/tumour of MES or DNA standard animals HEPES 5 mM reference w/waverage deviation treated 150/HEPES 1 699 938 1 398 072 6  nls-H 0.02 6819 100 5 860 709 6  150/5/HEPES 369 563 577 901 6* nls-H1 0.1 1 654 3132 145 147 6* nls-H1 0.02 4 031 000 1 007 996 6* 931 275 214 229 4  H 0.13 420 432 1 285 704 6  *mice put to sleep during the injection, bynarco-neuroleptanalgesia with an Imalgene + Rompun mixture (130 mg/kgketamine, 4 mg/kg xylazine, intraperitoneal route).

These results show that, in tumours which can be transfected by nakedDNA, the addition of peptide with low peptide/DNA ratios, withoutcombined cationic lipids, makes it possible to increase the expressionof the exogenic gene when compared with the naked DNA alone.

9 9 amino acids amino acid linear protein unknown 1 Lys Thr Pro Lys LysAla Lys Lys Pro 1 5 8 amino acids amino acid linear protein unknown 2Ala Thr Pro Ala Lys Lys Ala Ala 1 5 16 amino acids amino acid linearprotein unknown 3 Ala Thr Pro Ala Lys Lys Ala Ala Ala Thr Pro Ala LysLys Ala Ala 1 5 10 15 18 amino acids amino acid linear protein unknown 4Lys Thr Pro Lys Lys Ala Lys Lys Pro Lys Thr Pro Lys Lys Ala Lys 1 5 1015 Lys Pro 17 amino acids amino acid linear protein unknown 5 Ala ThrPro Lys Lys Ser Ala Lys Lys Thr Pro Lys Lys Ala Lys Lys 1 5 10 15 Pro 26amino acids amino acid linear protein unknown 6 Lys Lys Ala Lys Ser ProLys Lys Ala Lys Ala Ala Lys Pro Lys Lys 1 5 10 15 Ala Pro Lys Ser ProAla Lys Ala Lys Ala 20 25 18 amino acids amino acid linear proteinunknown 7 Ser Arg Ser Arg Tyr Tyr Arg Gln Arg Gln Arg Ser Arg Arg ArgArg 1 5 10 15 Arg Arg 21 amino acids amino acid linear protein unknown 8Arg Arg Arg Leu His Arg Ile His Arg Arg Gln His Arg Ser Cys Arg 1 5 1015 Arg Arg Lys Arg Arg 20 26 amino acids amino acid linear proteinunknown 9 Pro Lys Lys Lys Arg Lys Val Ala Lys Thr Pro Lys Lys Ala LysLys 1 5 10 15 Pro Lys Thr Pro Lys Lys Ala Lys Lys Pro 20 25

What is claimed is:
 1. A composition which is useful for thetransfection of an antisense nucleic acid, comprising a transfectionagent, an antisense nucleic acid, and a compound involved in thecondensation of the nucleic acid, wherein the compound comprises peptideunits as set forth in SEQ ID No. 1, and/or SEQ ID No. 2 repeatedcontinuously or non-continuously, wherein the number of peptide units isbetween 2 and 10, or the compound comprises an oligopeptide selectedfrom the group consisting of the oligopeptides as set forth in SEQ IDNo. 7 and SEQ ID No.
 8. 2. A composition which is useful for thetransfection of a therapeutic gene, comprising a transfection agent, thetherapeutic gene, and a compound involved in the condensation of thetherapeutic gene, wherein the compound comprises peptide units as setforth in SEQ ID NO. 1 or SEQ ID NO. 2 repeated continuously ornon-continuously, wherein the number of peptide units is between 2 and10, or the compound comprises an oligopeptide selected from the soupconsisting of the oligopeptides as set forth in SEQ ID NO. 7 and SEQ IDNO.
 8. 3. A composition according to claim 2, wherein the peptide unitsare separated from each other by connections, wherein the connectionsare biochemical compounds, amino acids or chemical compounds.
 4. Acomposition according to claim 3, wherein one of the connectionscomprises one or more amino acids.
 5. A composition according to claim2, wherein the compound is derived from histone H1.
 6. A compositionaccording to claim 5, wherein the compound is derived from theC-terminal domain of histone H1.
 7. A composition according to claim 5,wherein the compound is an oligopeptide selected from the groupconsisting of the oligopeptides as set forth in SEQ ID No.: 4, SEQ IDNo. 5, and SEQ ID No.
 6. 8. A composition according to claim 2, whereinthe compound is derived from the N-terminal domain of nucleoline.
 9. Acomposition according to claim 8, wherein the compound comprises apeptide as set forth in SEQ ID No.:
 3. 10. A composition according toclaim 2, wherein the compound possesses a ,sheet structure.
 11. Acomposition according to claim 2, wherein the compound is alsoassociated with a cell receptor ligand.
 12. A composition according toclaim 11, wherein the compound consists partly or totally of histone H1associated with a nuclear location signal sequence.
 13. A compositionaccording to claim 12, comprising the peptideProLysLysLysArgLysVal-βAlaLysThrProLysLysAlaLysLysProLysThrProLys-LysAlaLysLysPro(SEQ ID No.:9).
 14. A composition according to claim 2, wherein thecompound is also associated with a peptide of fusogenic type whichpromotes the cellular transfection of the composition.
 15. A compositionaccording to claim 2, wherein the compound is polyglycosylated,sulphonated, phosphorylated and/or grafted to complex sugars or to alipophilic agent.
 16. A composition according to claim 2, wherein thetransfection agent is a cationic polymer or a lipofectant.
 17. Acomposition according to claim 16, wherein the lipofectant is a lipidthat forms liposomes, furtive liposomes, immunoliposomes or targetliposomes.
 18. A composition according to claim 16, wherein the cationicpolymer is of general formula (I):

in which R is a hydrogen atom or a group of formula

n is an integer between 2 and 10; and p and q are integers, with the sump+q being such that the average molecular weight of the polymer isbetween 100 and 10⁷.
 19. A composition according to claim 16, whereinthe cationic polymer is selected from the group consisting ofpolyethyleneimine (PEI) and polypropyleneimine (PPI).
 20. A compositionaccording to claim 19, wherein the cationic polymer is selected from thegroup consisting of polyethyleneimine of average molecular weight 50,000(PEI50K) and polyethyleneimine of average molecular weight 800,000(PEI800K).
 21. A composition according to claim 16, wherein thelipofectant comprises a polyamine region of general formula (II)

in which m is an integer greater than or equal to 2 and n is an integergreater than or equal to 1, and wherein if n is greater than 1, then mis identical or different.
 22. A composition according to claim 21,wherein the polyamine region is spermine, thermine or one of theanalogues thereof that binds to the therapeutic gene.
 23. A compositionaccording to claim 21, wherein the lipofectant comprises a lipophilicregion represented by the general formula (IV)

in which X and X′ represent, independently of each other, an oxygenatom, a methylene group —(CH₂)_(q)— with q equal to 0,1, 2 or 3, or anamino group —NH— or —NR′— with R′ representing a Cl to C4 alkyl group, Yand Y′ represent, independently of each other, a methylene group, acarbonyl group or a C═S group, R₃, R₄ and R₅ represent, independently ofeach other, a hydrogen atom or a substituted or unsubstituted C₁ to C₄alkyl radical and p is between 0 and 5 inclusively, R₆ represents acholesterol derivative or an alkylamino group —NR₁R₂ with R₁ and R₂representing, independently of each other, a saturated or unsaturatedlinear or branched C₁₂ to C₂₂ aliphatic radical.
 24. A compositionaccording to claim 21, wherein the transfection agent comprises alipopolyamine selected from the group consisting ofdioctadecylamidoglycylspemrdne (DOGS), palmitoylphosphatidylethanolamine5-carboxyspermylamide (DPPES), 2,5-bis(3-aminopropylamino)pentyl(dioctadecylcarbamoylmethoxy)acetate,1,3-bis(3-aminopropylamino)2-propyl(dioctadecylcarbamoylmethoxy)acetate,{H₂N(CH₂)₃}₂N(CH₂)₄N{(CH₂)₃NH₂}(CH₂)₃NHCH₂COGlyN[(CH₂)₁₇—CH₃]₂,H₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COGlyN[(CH₂)₁₈]₂ andH₂N(CH₂)₃NH(CH₂)₄NH(CH₂)₃NHCH₂COArgN[(CH₂)₁₈].
 25. A compositionaccording to claim 2, wherein the transfection agent isdioctadecylamidoglycylspermine (DOGS).
 26. A composition according toclaim 2, wherein the therapeutic gene is encoded by a deoxyribonucleicacid.
 27. A composition according to claim 2, wherein the therapeuticgene is encoded by a ribonucleic acid.
 28. A composition according toclaim 2 wherein the therapeutic gene is encoded by a nucleic acid thatis chemically modified.
 29. A composition according to claim 2, whichfurther comprises a neutral lipid or lipids.
 30. A composition accordingto claim 29, wherein the neutral lipid or lipids are selected from thegroup consisting of synthetic lipids, natural lipids, zwitterioniclipids and lipids lacking ionic charge under physiological conditions.31. A composition according to claim 29, wherein the neutral lipid orlipids is selected from the group consisting ofdioleoylphosphatidylethanolamine (DOPE),oleoylpalmitoylphosphatidylethanolamine (POPE), di-stearoylphosphatidylethanolamine, -palmitoyl phosphatidylethanolamine,-myristoyl phosphatidylethanolamine, derivatives of di-stearoylphosphatidylethanolamine N-methylated 1 to 3 times, derivatives ofpalmitoyl phosphatidylethanolamine N-methylated 1 to 3 times,derivatives of myristoyl phosphatidylethanolamine N-methylated 1 to 3times, phosphatidylglycerol, diacylglycerol, glycosyldiacylglycerol,cerebroside, sphingolipid and asialoganglioside.
 32. A method oftransfer of a therapeutic gene comprising contacting a cell to betransfected with a composition according to claim 2, wherein thetherapeutic gene is transferred into the cell.
 33. The method of claim32, wherein the compound is an oligopeptide selected from the groupconsisting of the oligopeptides as depicted in: SEQ ID No.: 3, SEQ IDNo.: 4, SEQ ID No. 5, SEQ ID No. 6, SEQ ID No. 7, and SEQ ID No.
 8. 34.A composition according to claim 30, wherein the neutral lipid or lipidsare selected from the group consisting of galactocerebrosides,sphingomyelins, asialoGM1 and asialoGM2.
 35. The method of claim 32,wherein the compound comprises peptide units as set forth in SEQ ID No.:1 and/or SEQ ID No.:
 2. 36. A method of transfer of an antisense nucleicacid comprising contacting a cell to be transfected with a compositionaccording to claim 1, wherein the antisense nucleic acid is transferredinto the cell.